AB0065 N-cadherin is down-regulated by decoy receptor 3 in specifically rheumatoid synovial fibroblasts

Background Decoy receptor 3 (DcR3) is a secreted decoy tumour necrosis factor receptor and competitively binds and inhibits the TNF family including Fas-ligand, LIGHT, and TL1A. We previously reported that DcR3 overexpressed in rheumatoid synovial fibroblasts (RA-FLS) stimulated by TNFα protects the cells from Fas-induced apoptosis.1 We recently reported that DcR3 binds to TL1A expressed on RA-FLS resulting in the negative regulation of cell proliferation induced by inflammatory cytokines.2 Further, we newly revealed the gene expression profiles in RA-FLS regulated by DcR3 by using microarray data analysis.3 Among the genes in the profile, we demonstrated the possible involvement of tryptophan hydroxylase 1 down-regulated,4 interleukin 12B up-regulated by DcR35 and centrosomal protein 70 kDa6 in the pathogenesis of RA. The profiles also indicated that Cadherin 2/type 1/N-cadherin (CDH2) was up-regulated by DcR3 (fold change 1.93).3 CDH2 has been reported to be associated with cell attachment and migration,7 osteoblast differentiation,8 and the proliferation of RA-FLS.9 The hemophilic interaction of CDH2 suppresses the proliferation of RA-FLS through increasing the P27Kip1 that inhibit cell-cycle progression.9 Objectives In this study, we analysed CDH2 expression in RA-FLS stimulated with DcR3 in detail to reveal the involvement of CDH2 and DcR3 in the pathogenesis of RA. Methods Real-time polymerase chain reaction (real-time PCR). RA-FLS were stimulated with various concentration of DcR3-Fc or 1,000 ng/ml of IgG1, or left untreated in serum-free Opti-MEM for 12 hour. The relative expression levels of CDH2 mRNA were quantified by real-time PCR. Immunohistochemistry. Anti-CDH2 antibody was applied to frozen sections of synovial tissues from patients with RA or OA for overnight. After that, the expression of CDH2 protein was evaluated by immunohistochemical analysis. Results Real-time PCR demonstrated that DcR3-Fc significantly increased the expression of CDH2 mRNA in RA-FLS (104% with 10 ng/ml, 112% with 100 ng/ml, and 200% with 1000 ng/ml). Immunohistochemistry revealed that CDH2 was expressed more in the sublining layer of rheumatoid synovium than OA synovium. Conclusions In the gene expression profiles, we focused on CDH2 as the gene was highly up-regulated and belonged to major functional clustering categories; protein complex assembly, cell motility, regulation of transcription, cell membrane and glycosylation. In this study, we showed that the expression of CDH2 mRNA in RA-FLS was induced by DcR3 and that CDH2 was increased in the sublining layer of rheumatoid synovium. In this study, we demonstrated that DcR3 significantly induced CDH2 expression in RA-FLS. Considering the fact that CDH2 inhibits RA-FLS proliferation, DcR3 signalling may control the hyperplasia of RA synovium. References [1] Hayashi S, et al. Arthritis Rheum. 2007;56:1067–1074. [2] Takahashi M, et al. Int J Mol Med. 2011;28:423–427. [3] Fukuda K, et al. Int J Mol Med. 2013;32:910–916. [4] Maeda T, et al. Mol Med Rep. 2015;12:5191–5196. [5] Fukuda K, et al., Mol Med Rep. 2016;13:3647–3652. [6] Fukuda K, et al. Mod Rheumatol in press. [7] Akitaya T, et al. Dev Dyn. 1992;194:12–20. [8] Marie P. J Cell Physiol2002;190:297–305. [9] Nonomura Y, et al. J Rheumatol2009;36:698–705. Disclosure of Interest None declared